Methods for collecting and processing autografts, processed autografts, kits for collecting and transporting autografts, and tools for preparing autografts

ABSTRACT

The present invention is directed to methods for collecting and processing autografts, processed autografts, kits for collecting and transporting autografts, and tools for preparing autografts. It is also directed to autologous bone grafts, and methods of preparing them.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 17/101,294, filed on Nov. 23, 2020, now allowed, which is acontinuation of U.S. patent application Ser. No. 16/446,762, filed onJun. 20, 2019, now U.S. Pat. No. 10,856,545, issued on Dec. 8, 2020,which is a continuation of U.S. patent application Ser. No. 14/031,033,filed on Sep. 18, 2013, now U.S. Pat. No. 10,368,542, issued on Aug. 6,2019, which is a continuation of U.S. patent application Ser. No.12/825,033, filed on Jun. 28, 2010, now U.S. Pat. No. 8,541,233, issuedon Sep. 24, 2013, which claims priority to and the benefit of U.S.Provisional Application No. 61/220,779, filed on Jun. 26, 2009, each ofwhich are incorporated herein by reference for all purposes.

FIELD OF THE INVENTION

The present invention relates to methods for collecting and processingautografts, processed autografts, kits for collecting and transportingautografts, and tools for preparing autografts. The present inventionfurther relates to autologous bone grafts, and methods of preparingthem. The present invention also relates to autologous cranial boneflaps, and methods of preparing them.

BACKGROUND OF THE INVENTION

Approximately 30,000 traumatic brain injuries that require decompressivecraniectomy (e.g., removal of a cranial bone flap) occur in the UnitedStates every year. Decompressive craniectomies are used to treatpatients with massive brain swelling following either trauma or acutecerebral infarction to reduce intracranial pressure. Intracranialsubdural grid monitoring is used as a diagnostic technique in patientswith intractable partial epilepsy to identify an epileptogenic focus forsurgical intervention, which also requires the removal of a bone flapfrom a patient. In some patients surgical removal of a cranial bone flapis necessary to provide access to a tumor or an aneurysm. Immediatelyfollowing such surgeries, particularly in decompressive craniectomies,the surgical defect may be left open for some period of time, in orderto permit swelling and inflammation of brain tissue to recede. The woundmay be temporarily covered by a helmet and/or bandages as the patientrecovers.

However at some time after such surgical procedures (i.e., decompressivecraniectomy, or intracranial electroencephalography, among others) inwhich bone is explanted from a patient's skull, it becomes necessary fora surgeon to provide a structural replacement that will act as aprotective barrier for the patient's brain in the long term. Severalfactors are considered by surgeons when selecting a replacementincluding: (1) tissue reactions including immunogenic responses, metaltoxicities reactions, and others, (2) fit, (3) potential for infection,and (4) aesthetic results. Surgeons may store the patient's own cranialbone flap for future re-implantation, or they may choose from severalsynthetic replacements (i.e., poly-methyl-methacrylate (PMMA) implants,hydroxyapatite implants, or titanium implants, among others) forreconstruction.

Re-implanting the patient's own bone is considered to be the best optionby the majority of surgeons. In contrast to synthetic replacements,which may cause immunological reactions or other tissue reactions, anautologous cranial bone flap does not pose this risk as it is thepatient's own tissue. While synthetic replacements may requiresignificant manipulation to create a good fit and to produce a goodcosmetic effect, an autologous bone flap matches the contour of thepatient's defect and provides a more natural appearance after healing.In fact, synthetic implants have a number of disadvantages: (1) theirpreparation can be difficult, time-consuming, and expensive, (2)clinical and aesthetic outcomes can be unpredictable, (3) they do notpermit osteointegration, (4) resorption of host bone may occur at theimplant site requiring additional reconstructive surgery, and (5)immunogenic responses to the synthetic materials are a possibility. Theproblems associated with synthetic implants are increased, when thedefect being repaired is relatively large. Despite their drawbacks, manyhospitals still choose synthetic implants, because (1) they do not havethe facilities to safely store/process bone flaps, or (2) they view theuse of synthetic implants as less likely to result in an infection atthe surgical defect.

The rate of infection at a surgical defect closed with a patient'sre-implanted bone flap can be relatively high (estimates have beenplaced between 2% and 20%).

Surgeons have tried a variety of methods to minimize the risk ofinfection associated with the use of autologous bone flaps to closedefects, including: ethylene oxide and/or steam sterilization, freezing,boiling in saline, immersion in hydrogen peroxide solution, and storagein the patient by surgical intra-abdominal placement. These methods havemet with only limited success. Still further, many surgical facilitiesdo not have access to resources that would permit them to implementmethods of treating bone flaps (for example, many facilities do not havefreezers dedicated to autograft tissue storage). Also, some of themethods create additional problems or potential liability for thesurgical facilities (for example, if tissue freezers are available, theymust be properly overseen and maintained to prevent cross-contaminationof stored tissues and unintended thawing of the freezer contents; or ifintra-abdominal placement is used for storage of a bone flap, theadditional surgery may increase the likelihood of surgical complicationsin the patient).

When an autologous bone flap does become contaminated by an infection,the surgeon is often forced to remove and discard it, and to fashion asynthetic replacement. Alternatively, the surgeon may treat thecontaminated bone flap with steam sterilization or in situ antibioticirrigation. Neither is an ideal solution. For example, in situantibiotic irrigation involves removing the contaminated bone flap,scrubbing it with povidone-iodine solution, and soaking in a hydrogenperoxide solution, while the wound is debrided. The bone flap is thenre-implanted and an irrigation system is installed with it, andantibiotic medications are infused through the system for several days.The in situ antibiotic irrigation requires a complex set-up andtreatment regimen, and an extended hospital stay. It is also ineffectivein some situations (i.e., where there is sinus involvement, or when thedefect is at the skull base, among others), and it has the potential forcausing undesirable immunogenic reactions.

Using methods and autografts of the present invention, hospitals may beprovided with autologous grafts that are less expensive than syntheticimplants, aesthetically and clinically satisfactory, well-fitting andeasy to use, and safe.

SUMMARY OF THE INVENTION

The present invention provides methods for collecting and processingautografts (e.g., autologous tissue grafts), autografts, kits forprocuring and transporting autografts, and tools for preparingautografts.

The method of the present invention may comprise a method for collectingand processing autologous tissue for re-implantation in a patientcomprising surgically removing a piece of tissue from a patient;treating the tissue with one or more treatment solutions; processing thetreated tissue; and re-implanting the processed tissue into the patient.The method of the present invention may also consist of surgicallyremoving a piece of tissue from a patient; treating the tissue with oneor more treatment solutions; processing the treated tissue; andre-implanting the processed tissue into the patient.

Certain aspects of the present invention are directed to methods forcollecting and processing autologous tissue for re-implantation in apatient. Some methods of the present invention may include: (1)surgically removing a piece of a tissue from a patient to obtain acollected tissue; (2) pre-treating the collected tissue with at leastone treatment composition to produce a pre-treated tissue; (3) shippingthe pre-treated tissue to a tissue processing facility; (4) processingthe pre-treated tissue to produce a processed tissue; (5) shipping theprocessed tissue to a surgical facility; and (6) re-implanting theprocessed tissue into the patient from whom the piece of tissue wassurgically collected. Other methods of the present invention may consistessentially of (1) surgically removing a piece of a tissue from apatient to obtain a collected tissue; (2) pre-treating the collectedtissue with at least one treatment composition to produce a pre-treatedtissue; (3) shipping the pre-treated tissue to a tissue processingfacility; (4) processing the pre-treated tissue to produce a processedtissue; (5) shipping the processed tissue to a surgical facility; and(6) re-implanting the processed tissue into the patient from whom thepiece of tissue was surgically collected.

In some aspects of the invention, the tissue may be removed from asubject. The subject may be a patient. The patient may be an animal. Theanimal may be a mammal. The mammal may be a human, a cow, a pig, a goat,a dog, a horse, or a sheep.

In some aspects of the present invention, a tissue collected andprocessed using methods of the present invention may include cartilage,bone, or soft tissue (i.e., tendons, among others). Certain aspects ofthe present invention are directed to methods for collecting andprocessing autologous bone tissue for re-implantation in a patient, andin some methods of the present invention, the autologous bone that iscollected and processed is autologous cranial bone such as a cranialbone flap. For example, a craniectomy is performed to remove the cranialbone flap. The bone flap may be processed for re-implantation such as ina future cranioplasty. A collected tissue may be contaminated with atleast one of debris, a bacterium, a single-celled eukaryote (i.e., afungus or amoeba, among others) or a virus.

Besides cranial bone flaps, other types of tissues including but notlimited to facial bone, tibial bone, femoral bone, bones of theextremities, periprosthetic bone, ligaments, tendons, and muscle, amongothers, may be removed from a patient, processed, treated, andre-implanted. Facial bones may include the mandible, frontal bone,parietal bone, sphenoid bone, temporal bone, nasal bone, among others.Bones of the extremities may include bones of the hands, feet, arms, andothers. Periprosthetic fractures may be difficult to fix. The presentprocess provides a method of cleaning the bone with the implant attachedto it as a way of fixing periprosthetic.

A surgeon would prefer to re-implant the tissue into a patient, ratherthan replace it with an allograft or synthetic graft. However, existingmethods for preparing such tissues for re-implantation may present anunacceptable level of risk for post-operative infection. For example,tissues such as bones may be damaged due to trauma, infection, ordisease (i.e., cancer, among others), and a surgeon may wish tore-implant the facial bone, but might be unwilling to do so knowing thepossible risks of infection using known methods. Depending on the natureof a facial trauma there may be dirt or debris in the wound, and theseneed to be removed to reduce the likelihood of infection before damagedfacial bone can be re-implanted. In some cases, a soft tissue (i.e.,ligament, tendon, muscle or fascia, among others) may be infected or beunclean due to trauma, and a surgeon may need to remove the tissue. Thesurgeon might prefer to re-implant the soft tissue, but be unable to doso using existing methods without a serious risk of infection.Similarly, a surgeon may need to remove some bone, when a patientpresents with a periprosthetic fracture (i.e., a fracture of a bonesurrounding a knee or hip replacement, among others), and the surgeonmay wish to re-implant the bone (which may be attached to part of aprosthesis), but current methods of preparing the removed bone may notbe sufficiently rigorous for a surgeon to accept this as a safealternative.

In some aspects of the present invention, a collected tissue is treatedwith at least one treatment composition before being shipped to a tissueprocessing facility. In certain aspects of the present invention, amethod for collecting and processing an autologous tissue may include atleast one cleaning step and at least one rinsing step. In some aspectsof the present invention, a collected tissue is processed usingAllowash® treatment (a proprietary tissue cleansing process of LifeNetHealth, Virginia Beach, Va.) at a tissue processing facility.

The tissue may be sterilized by irradiation. The tissue may beirradiated with gamma rays. Other methods of sterilizing the tissueinclude but are not limited to using supercritical carbon dioxide,electron beam, and ethylene dioxide. The tissue may be sterilized priorto or after processing. The tissue may be sterilized prior to packagingor after being packaged for shipment.

The tissue processing facility may be the surgical facility where thetissue is removed and/or re-implanted or a separate surgical facility.The tissue processing facility also may be a facility separate from thesurgical facility.

The tissue may be packaged for shipment to the processing facility forprocessing. After processing, the tissue may be packaged for shipment tothe surgical facility for re-implantation. Prior to or after packaging,the processed tissue may be irradiated for sterilization purposes beforeshipment to the proper facility.

In one aspect of the invention, during pre-treatment or afterpre-treatment of the tissue, one or more bactericidal agent and/or oneor more preservation agent or plasticizer may be added to the tissueprior to processing or shipment to a processing facility for processing.In another aspect of the invention, during processing or afterprocessing of the tissue, one or more bactericidal agent and/or one ormore preservation agent or plasticizer may be added to the tissue forstorage and/or shipment to a surgical facility for re-implantation.

In certain aspects of the present invention, an instrument insertionsite is added to a collected tissue for ease in maneuvering the tissue.In some aspects of the present invention, at least one of a compositionincluding an allograft tissue, a composition including a synthetic graftmaterial, or a biocompatible adhesive is added to an autograft(autologous tissue) during processing. In certain aspects of the presentinvention, a processed tissue that has been packaged is gamma irradiatedbefore re-implantation. In some aspects of the present invention apackaged processed tissue has a sterility assurance level (SAL) of atleast about 10−⁶. In certain aspects of the present invention, aprocessed tissue is stored for between about 3 days to 5 days, 3 daysand 1 year before it is re-implanted in the patient. In some instances,the tissue is processed and stored at the surgical facility or thehospital and re-implanted. As an example, for brain tumor patients orfor injuries resulting from battle field trauma, the processed tissuesmay be re-implanted in the patient in about 3 to 5 days and thereforewill not be shipped or stored at a tissue processing facility but willbe processed, treated, and stored at the surgical facility or hospitaland re-implanted.

In some methods of the present invention, a status report regarding anautologous tissue is entered into a computer accessible informationsystem at each step in the method. In certain methods of the presentinvention, a tracking device is kept with a tissue throughout themethod.

Some aspects of the present invention are directed to autologous tissueprocurement kits for use in collecting a piece of a tissue from apatient. In certain aspects of the present invention, the kit mayinclude (1) an instruction sheet containing at least a contact phonenumber for a tissue processing facility, a description of autologoustissue collection kit use, and a description of a method of packaging acollected tissue; (2) at least one label including a space for apatient's identifying information to be entered; (3) a sealable tissuecontainer; (4) a shipping container; and (5) at least one of a treatmentcomposition, and (6) an electronic storage device (e.g., a microchip)for storing information electronically. Some kits of the presentinvention may include a tool for creating an instrument insertion sitein a tissue, or a surgical instrument for re-implanting an autografthaving an instrument insertion site.

As an example, a kit of the present invention may include (1) acontainer for holding the tissue after removal from patient at thesurgical facility; (2) a treatment solution for cleaning and/ordisinfecting the tissue at the surgical facility; (3) temporary storageand shipment solution; (4) a device for adding an instrument insertionsite to the tissue for ease of maneuvering the tissue; (5) a microchipfor tracking the tissue; (6) a container for storage at the surgicalfacility and/or shipment to the processing facility; and (7) a coolercomprising a compartment for storing the tissue and other compartmentsfor storing other accessories. The kit may include more than one of eachof these components. The present invention also provides methods forusing the kit disclosed herein for procuring and processing autologoustissues for re-implantation in a patient.

Certain aspects of the present invention are directed to autologous bonegrafts for re-implantation into the patient. The re-implanted bone graftmay include a piece of bone that is autologous to the recipient, and thepiece of bone may include an instrument insertion site that is capableof securely coupling to a surgical placement instrument and to at leastone of (I) a solution injector and (2) a solution evacuator. In someaspects of the present invention, the autologous bone graft furtherincludes at least one of an allograft tissue and a synthetic implantmaterial. In certain aspects of the present invention, the autologousbone graft is sterile. Some autologous bone grafts of the presentinvention may be prepared from a tissue that had been contaminated witha bacteria or a virus. Certain aspects of the present invention aredirected to packaged autologous bone grafts including a packagecontaining an autologous bone graft having a sterility assurance levelof at least about 10−⁶.

The method of the present invention also may be performed with organssurgically removed from a subject or a patient. As an example, the organmay be surgically removed from a subject, pretreated before processing,processed, and re-implanted into the subject. The organ may be packagedfor shipping to a processing facility. The processed organ may be storedafter processing. The processed organ may be packaged and shipped to asurgical facility for re-implantation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. FIG. 1A is a picture of the bone flap of Example Ibefore processing. FIG. 1B is a picture of the bone flap of Example Iafter processing.

FIGS. 2A and 2B. FIG. 2A is a picture of the bone flap of Example 2before processing. FIG. 2B is a picture of the bone flap of Example 2after processing.

FIGS. 3A and 3B. FIG. 3A is a picture of the bone flap of Example 3before processing. FIG. 3B is a picture of the bone flap of Example 3after processing.

DESCRIPTION OF THE INVENTION

Some aspects of the present invention are directed to methods forsurgically removing a tissue from a patient to obtain a tissue,pre-treating the tissue prior to processing, and processing the tissuefor re-implantation in a patient.

Certain methods of the present invention may include the steps of: (1)surgically removing and collecting a piece of a tissue from a patient toproduce a collected tissue; (2) treating (or pre-treating) the tissue ata surgical facility prior to processing at a tissue processing facility;(3) shipping the collected tissue to a tissue processing facility; (4)processing the collected tissue to produce a processed tissue; (5)shipping the processed tissue to a surgical facility; and (6)re-implanting the processed tissue into the patient from whom the pieceof tissue was surgically collected.

In some instances, the surgically removed tissue may need to bere-implanted a few days after surgical removal from the patient. Thetissue may be processed and stored at the surgical facility forre-implantation in a few days, for example 3 to 5 days, after surgicalremoval. Accordingly, the tissue processing and storage facility may bethe surgical facility or hospital. Thus, certain methods of the presentinvention may include the steps of: (1) surgically removing andcollecting a piece of a tissue from a patient to produce a collectedtissue; (2) treating (or pre-treating) the collected tissue at asurgical facility; (3) processing the collected tissue at the surgicalfacility; and (4) re-implanting the processed tissue into the patientfrom whom the piece of tissue was surgically collected. A collectedtissue processed at a surgical facility may optionally be stored at asurgical facility or at an external storage facility before beingre-implanted, in some aspects of the present invention. In certainmethods of the present invention, a collected tissue processed at asurgical facility may be re-implanted in the patient within about 3 daysto about 5 days (i.e., where tissue is removed to access a brain tumoror during surgical intervention of a battlefield trauma).

In some methods of the present invention, the collected and processedtissue includes at least one of cartilage, bone, and a soft tissue fromthe patient. The tissue includes bone from the patient, in some aspectsof the present invention. In certain aspects, the bone includes cranialbone, facial bone, tibial bone, femoral bone, bone from the extremities(i.e., the hands, feet, and arms), and bone from a periprostheticfracture. The bone tissue includes cranial bone from the patient incertain aspects. In some aspects, the bone tissue includes a cranialbone flap from the patient. Soft tissue may include but are not limitedto tendon, ligament, fascia, and muscle. In certain aspects of thepresent invention, the tissue collected and processed includes (1) boneand soft tissue (i.e., tendon) from the patient, or (2) bone andarticular cartilage from the patient. A tissue that is collected fromthe patient for processing may be contaminated with at least one ofdebris, a bacterium, a single-celled eukaryote (i.e., a fungus, amongothers) and a virus. Accordingly, the tissue may need to be treated andprocessed prior to re-implantation in the patient.

In certain aspects of the present invention, a method of collecting andprocessing autologous tissue includes providing a tissue collection kitthat is used in the surgical removal of tissue from the patient. In someaspects, the tissue may be a cranial bone flap that has been surgicallyremoved during a decompressive craniectomy, an intracranialelectroencephalography procedure, a brain tumor surgery, or a brainaneurysm surgery, among others. In certain methods of the presentinvention, a cranial bone flap may be collected from the patient duringa decompressive craniectomy, or after infection of a part of thepatient's skull.

In some aspects of the present invention, a tissue that has beencollected from the patient may be pre-treated with one or more treatmentcompositions before it is packaged and shipped to a tissue processingfacility for processing. The treatment composition may be antimicrobialsolution, an antibiotic solution, a plasticizer solution, sterile water,sterile saline, or a detergent solution, among others. The treatmentsolution may also comprise the proprietary Allowash® solution technologyof LifeNet Health, Virginia Beach, Va. The plasticizer solution maycontain glycerol. The treatment composition may also include adisinfectant and/or a plasticizer. In some aspects, a treatmentcomposition may include hydrogen peroxide. In certain aspects, atreatment composition may include a plasticizer, such as glycerol. Inother embodiments, the treatment may comprise the use of a bactericidaland/or a virucidal agent. In certain aspects of the present invention, atreatment composition may include antibiotic, plasticizer, water, abuffer, a detergent, an enzyme, a bioactive factor, or a combinationthereof. A treatment composition may be a treatment solution, in certainaspects of the present invention. In some aspects of the presentinvention, a treatment composition may reduce the bioburden of acollected tissue. A collected tissue may be placed in a treatmentcontainer with a treatment and/or short term or temporary storagecomposition before it is shipped to a processing facility in certainaspects of the present invention. The short term or temporary storagecomposition may include hydrogen peroxide and/or a plasticizer in someaspects of the present invention.

During pre-treatment or after pre-treatment of the tissue, one or morebactericidal agent and/or one or more preservation agent or plasticizermay be added to the tissue prior to processing or prior to shipping to aprocessing facility for processing. Moreover, during processing or afterprocessing of the tissue, one or more bactericidal agent and/or one ormore preservation agent or plasticizer may be added to the tissue forstorage and/or shipment to a surgical facility for re-implantation.

The present invention also provides a container for holding the tissue.The container may have one or more openings or ports for liquids toenter and exit the container and an opening for placing the tissue intothe container. The container may be deformable, nondeformable, or acombination thereof in which the container has a nondeformable exteriorand a deformable interior. The container may be made of material thatenables it to be deformable, or nondeformable or a combination thereof.The container may have an inlet opening and an outlet opening forrinsing solutions, treatment solutions, and storage solutions to enterand exit. The opening on the container for placing the tissue into thecontainer or for removing the tissue from the container may be closedwith a zipper or with a clamp. The process of cleaning, disinfecting,treating, storing, and/or shipping of the tissue may all be performed inthe container of the present invention. The container may be a bag ormay contain a bag with the features described above for the container.

In some aspects of the present invention, a collected tissue is packagedby a method including placing it into a shipping container with atracking device and/or labeling the shipping container with at least apatient identification number. In certain methods of the presentinvention, a collected tissue is packaged by a method including, placingthe collected tissue in a container and sealing the container, placingthe sealed container into a shipping container, and adding dry ice orwet ice between the sealed container and the shipping container. In someaspects, the shipping container includes an insulating insert. Incertain aspects, the shipping container includes a cooler, and in someaspects the cooler includes a temperature regulating device (i.e., arefrigeration unit). A collected tissue may be shipped to atissueprocessing facility at room temperature, on dry ice, or on wet ice, incertain methods of the present invention.

In certain aspects of the present invention, a tissue is surgicallyremoved from the patient, packaged, and shipped to a tissue processingfacility, where it is unpacked and undergoes processing. In some methodsof the present invention, processing (e.g., at the tissue processingfacility or the surgical facility) may include at least one ofdebridement, decontamination, and removal of cellular elements, amongothers. In some aspects of the present invention, processing causes atissue to have a reduced bioburden. In certain aspects, processing mayinclude the use of at least one of a detergent, an antimicrobial, anantibacterial, an antibiotic, and an alcohol to affect removal ofassociated tissues (such as, as bone marrow, lipids, and cellularelements in a bone tissue, among others) and/or contaminants (such as,bacteria and viruses, among others). In some aspects of the presentinvention, a tissue obtained from a patient may be processed with asolution for removing lipids and blood cells from the tissue such asacetone, with a solution inducing osmotic shock for removing nucleicacids from the tissue, and/or with an oxidizing agent, such as hydrogenperoxide for removing and/or inactivating immunogenic structures andmicroorganisms. The tissue may also be processed by treating withorganic solvents.

In some aspects, processing includes cleaning the tissue. The tissuethat was removed from the patient may be cleaned using methods anddevices known in the art. In certain aspects of the present invention, atissue obtained from the patient may be processed using proprietaryAllowash® technology of LifeNet Health, Virginia Beach, Va. Processingthat involves the use of Allowash® may or may not include debridement.If debridement and Allowash®, or other processing techniques, are usedwith one another, the debridement may occur either before or after thetissue is processed with the Allowash® or processing solutions.Exemplary methods, compositions, and devices for processing includethose disclosed in U.S. Pat. Nos. 6,942,961; 6,482,584; 6,024,735;5,977,432; 5,977,034; 5,976,104; 5,846,484; 5,820,581; 5,797,871;5,556,379; 7,648,676 and 7,585,461; U.S. Publication Nos. 2008/0188939;2005/0100862;

2005/0096742; and 2004/0037735; and International Publication No. WO2004/075935, which are hereby incorporated by reference in theirentirety.

In some aspects of the present invention, the processing, at aprocessing facility or a surgical facility, includes at least onecleaning step and at least one rinsing step. A rinsing step may permitthe removal of any residual processing components that may cause anegative tissue reaction in the patient. Processing may includedecellularization or devitalization of a tissue, as is known in the art,in certain aspects of the present invention. Exemplary decellularizationand devitalization methods include those disclosed in U.S. Pat. Nos.7,338,757; 6,743,574; and 6,734,018, among others. Several autograftsmay be processed by one method at the same time, in some methods of thepresent invention. In certain aspects of the present invention,processing may include using, for example, centrifugation, sonication,application of a vacuum, or a combination thereof.

In certain methods of the present invention, bone tissue collected fromthe patient may be demineralized as part of its processing at a tissueprocessing facility. In some aspects, the bone tissue may bedemineralized by exposure to dilute hydrochloric acid, as is known inthe art. Any demineralization processes known in the art, may be used toprepare demineralized bone for re-implantation into the patient.Exemplary demineralization processes are described in U.S. Pat. Nos.6,830,763; 6,534,095; 6,305,379; 6,189,537; 5,531,791; and 5,275,954,which are hereby incorporated by reference in their entirety. In certainmethods of the present invention, processing of a bone tissue mayinclude a perforation method. In some aspects of the present invention,processing at a tissue processing facility yields a cleaned,demineralized cranial bone flap or a cleaned, perforated cranial boneflap.

In some aspects of the present invention, processing, at a processingfacility or a surgical facility, may include treating the tissue with asolution including glycerol (i.e., proprietary Preservon® treatments ofLifeNet Health, Virginia Beach, Va.). In certain aspects of the presentinvention, tissues collected from the patient may be processed asdescribed in U.S. Pat. Nos. 7,063,726; 6,569,200; 6,544,289; and6,293,970, which are hereby incorporated by reference in their entirety.

In certain aspects of the present invention, processing may includeadding an instrument insertion site in a tissue collected from thepatient. An instrument insertion site may be used to connect a syringeor a pump (i.e., a vacuum pump, among others) to a tissue in someaspects of the present invention. Thus, a processing/treatment solutionor a rinsing solution may be applied to a tissue by flushing it throughthe instrument insertion site, in certain aspects of the presentinvention. Rinsing of an autograft may be done in a surgical facility bycoupling a syringe of or a pump for rinsing solution to an instrumentinsertion site in the autograft and flushing the autograft, in someaspects of the present invention. In certain aspects of the presentinvention, a processed tissue may be packaged in a syringe-adaptedcontainer, and a syringe of rinsing solution may be coupled to thesyringe-adapter in a surgery facility where it is rinsed.

Alternatively, in some aspects of the present invention, aprocessing/treatment solution, a storage solution, or a rinsing solutionmay be drawn through an autograft by methods employing a syringe or apump (e.g., vacuum) coupled to the instrument insertion site of theautograft.

In some aspects of the present invention, processing, at a processingfacility or a surgical facility, may include adding bioactive factors tothe tissue that was collected from the patient. Bioactive factors arefactors that regulate growth, proliferation, differentiation, migration,and maturation of cells. In some aspects of the present invention,bioactive factors may be osteoinductive factors, chondrogenic factors,cytokines, mitogenic factors, chemotactic factors, transforming growthfactors (TGFs), fibroblast growth factors (FGFs), angiogenic factors,insulin-like growth factors (IGFs), platelet-derived growth factors(PDGFs), epidermal growth factors (EGFs), vascular endothelial growthfactors (VEGFs), nerve growth factor (NGFs), neurotrophins, bonemorphogenetic proteins (BMPs), osteogenin, osteopontin, osteocalcin,cementum attachment protein, erythropoietin, thrombopoietin, tumornecrosis factor (TNF), interferon, colony stimulating factors (CSFs), orinterleukins, among others. The bioactive factor may be rhBMP2(recombinant human BMP2), rhBMP4 (recombinant human BMP4), rhBMP7(recombinant human BMP7), or rhPDGF (recombinant human platelet-derivedgrowth factor), among others, in some aspects of the present invention.In certain aspects of the present invention, a bioactive factor may bean enzymatically treated or modified bioactive factor. For example, thebioactive factor may have been treated with an enzyme to have a modifiedactivity or treated chemically to have a modified activity.

In certain aspects of the present invention, processing, at a processingfacility or a surgical facility, may include applying a compositionincluding at least one of an allograft tissue and a synthetic graftmaterial to the autologous tissue to aid in wound healing, when it isre-implanted in the patient. In some aspects of the present invention, apaste, a gel, a putty, or a sponge may be applied to a surface and/oredge of a processed autologous tissue to improve healing and union, whenthe tissue is re-implanted in the patient. In certain aspects of thepresent invention, when the autograft is a cranial bone flap, a sponge(i.e., a collagen sponge) may be applied to cover the bone flap to aidin reconstruction of the patient's periosteum and to improve theremodeling rate of the bone flap. Sponges, gels, pastes, and puttiesused in some aspects of the present invention may include an allograftmaterial or a synthetic graft material (i.e., polymers, calciumphosphate or hydroxyapatite, among others). For example, sponges andputties including demineralized allograft bone may be used in someaspects of the present invention.

In some methods of the present invention, after an autograft isprocessed, it is packaged before it is re-implanted in the patient. Incertain aspects of the present invention, a packaged processed tissue isgamma irradiated before shipping to the surgical facility. Irradiationof a packaged processed tissue may be performed at the facility where atissue is processed or at a separate facility. In some aspects of thepresent invention, a packaged processed tissue is subjected to gammairradiation (i.e., about 18 kGy to about 58 kGy minimum absorbed dose)at a low temperature (i.e., −20° C. to −50° C.). In certain aspects ofthe present invention, the gamma irradiation process may be validatednot to damage biological or structural capabilities of a tissue graft.In some aspects, a packaged processed tissue of the present inventionhas a sterility assurance level (SAL) of at least about 10−⁶

After processing, the tissue may be sterilized by irradiation with about18 KGy to about 25 KGy. It may be necessary to irradiate at a low dosageso as not to compromise the biomechanical properties of the tissue. Thetissue may be sterilized with gamma irradiation. In some aspects of thepresent invention, a tissue obtained from a patient may be sterilizedusing electron beam, supercritical CO₂, or ethylene oxide methods, amongothers.

In other embodiments of the present invention, the processed tissue maybe packaged in a storage solution. The processed tissue may be stored inthe storage solution either before or after it is sterlized, for examplethrough gamma irradiation. In certain aspects of the invention, theprocessed tissue is stored in a storage solution and subsequentlysterilized in the storage solution. Examples of storage solutionsinclude, but are not limited to, solutions including glycerol (i.e.,proprietary Preservon® treatments of LifeNet Health, Virginia Beach,Va.), or other polyol, high concentration salt solution, for example,sodium chloride, as disclosed in U.S. Pat. No. 6,739,112 (incorporatedby reference), and DMSO, as disclosed in U.S. Pat. No. 6,946,098(incorporated by reference) or combinations thereof.

In some methods of the present invention, packaging of a processedtissue includes placing the processed tissue into a syringe-adaptedcontainer. The syringe-adapted container may then be placed into ashipping container, in certain methods. In some aspects of the presentinvention, a shipping container may include a compartment for holding acontainer containing a processed tissue and a compartment for holding agraft instruction sheet, and a shipping container may, optionally,include additional compartments. In certain aspects of the presentinvention, at least one of (1) a microchip, (2) a surgical placementinstrument, (3) a biocompatible adhesive (i.e., a fibrin-based adhesiveor collagen gel, among others), (4) a composition including syntheticgraft materials (i.e., polymers, hydroxyapatite, or calcium phosphateamong others) for aiding in wound healing and union, and (5) acomposition including an allograft tissue (i.e., collagen, cells, anddemineralized bone, among others) for aiding in wound healing and unionmay also be placed into a shipping container before a packaged processedautologous tissue is shipped to a surgical facility. In certain aspects,the shipping container includes a cooler, and in some aspects the coolerincludes a temperature regulating device (i.e., a refrigeration unit).

In some methods of the present invention, a swab culture may be preparedfrom at least one of the collected tissue, the pre-treated tissue, andthe processed tissue. A tissue is swabbed using methods known in theart, and the swab is placed in contact with at least one culture mediumunder various culture conditions, so that any bacteria, fungi, or othersingle-celled organisms present may grow, be detected, and beidentified. Swab cultures may be prepared using thioglycolate and/ortrypticase soy broth media, among others, in certain aspects of thepresent invention. Swab cultures may be grown at two or moretemperatures, and for up to about 14 days, in some methods of thepresent invention. When a tissue is swab tested before processing, theswab culture may be performed by a surgical facility where the tissue iscollected from the patient and/or at a tissue processing facility. Swabcultures may permit a processing facility to monitor the effectivenessof its processing. A swab culture test of a processed tissue may yieldno microbial growth (i.e., bacterial growth) in some aspects of thepresent invention.

In certain aspects, a swab culture of a processed tissue may yield nomicrobial growth, even where the tissue was contaminated beforeprocessing.

In certain methods of the present invention, a processed tissue issubjected to a quality assurance review at the tissue processingfacility before it is shipped to a surgical facility. Those responsiblefor quality assurance may release an autologous tissue only afterreviewing records to ensure all standards were met during processing, insome aspects of the present invention. In certain aspects of the presentinvention, a donor records coordinator may ensure final documentation iscomplete before a processed tissue is sent to a surgical facility to bere-implanted in the patient from which it was originally collected. Insome methods of the present invention, a packaged processed autograft isshipped to a surgical facility in an organic, non-toxic and fullybiodegradable shipping container with a prepaid return label, andinstructions for use. The shipping container may contain at least one ofa gel, a putty, a paste, a sponge, and a biocompatible adhesive that canbe employed at the defect site during re-implantation of the autograft,so that it may aid in at least one of wound healing, union of the graftwith tissue at the implant site, and keeping the graft in place untilunion of the tissues occurs.

In certain aspects of the present invention, a method of collecting andprocessing autologous tissue, including surgical collection, andoptionally, shipping to and processing at a tissue processing facility,and shipping of the processed tissue to a surgical facility, requiresless than two weeks. In some methods of the present invention aprocessed tissue is stored at a tissue processing facility, a surgicalfacility, or an independent tissue storage facility for between about 3days and 1 year before it is re-implanted. When a cranial bone flap isremoved from a patient with a traumatic brain injury (TBI), it can takemore than six months before that patient is in a condition to have thebone flap re-implanted. Thus, it may be necessary to store a bone graftfor some time before it is re-implanted in the patient. In some cases acranial bone flap created during brain cancer and aneurysm surgery maybe re-implanted in the patient in as little as 3 to 4 days. In someaspects of the present invention, storage of a processed tissue occursin a facility that may be validated annually, have restricted access,have 24-hour monitoring, have internal and/or external alarms, and havea natural disaster recovery program. In certain aspects of the presentinvention, a processed tissue may be stored in a freezer, and thefreezer may be calibrated annually. In some aspects of the presentinvention, a processed tissue may be stored at a temperature betweenabout −40° C. and about −80° C., or between about −20° C. and about −40°C.

In certain methods of the present invention, a tissue that has undergoneprocessing may be seeded with a plurality of viable cells before it isre-implanted into the patient from whom the tissue was originallycollected. In some methods of the present invention, the cells seededonto the tissue may be allogenic or autogenic to the patient that thetissue will be re-implanted into. Cells may be added to a processedtissue using methods known in the art, in some aspects of the presentinvention. A tissue collected from a patient that has been processed (asdiscussed above) may have cultured cells derived from the patient oranother individual of the same species added to it before it isre-implanted in the patient, in certain aspects of the presentinvention. In some aspects of the present invention, bone tissue (i.e.,a cranial bone flap) taken from a patient, and then processed, may havecultured osteoblasts derived from the same patient or another individualof the same species added to it before it is re-implanted in thepatient. In some aspects of the present invention processed bone tissueto which cells are added may have been demineralized during processing.Methods for adding cells to a demineralized bone tissue known in the artmay be use, and exemplary methods are disclosed in U.S. Pat. No.7,494,811, among others. In certain aspects of the present invention, atleast one of osteoblasts, pre-osteoblasts, osteocytes, chondrocytes,chondroblast, periosteal cells, myoblasts, endothelial cells,mesenchymal cells, and fibroblasts may be added to a processed tissuebefore it is re-implanted in the patient. In some aspects of the presentinvention the osteoblasts, pre-osteoblasts, chondrocytes, chondroblasts,periosteal cells, myoblasts, endothelial cells, mesenchymal cells, orfibroblasts are cultured from cells derived from a donor or from thesame patient receiving the processed tissue autograft.

In some methods of the present invention, a status report is enteredinto a computer accessible information system at each step (i.e.,shipping step, processing step, among others) in the method. In certainmethods of the present invention a tracking device is kept near (i.e.,within 5 feet) the tissue throughout the method. In some aspects of thepresent invention, the tracking device includes a microchip. Thetracking device may be embedded in the tissue in the form of amicrochip. In certain aspects of the present invention, the trackingdevice includes a thumb drive. The tracking device has at least apatient identification number stored on it, in some aspects of thepresent invention. In certain aspects of the present invention,processing conditions used with an autologous tissue graft may be storedon the tracking system. Where the tracking system includes a microchip,the microchip may be scanned and information updated at each step incertain methods of the present invention. In some aspects of the presentinvention, information stored on the microchip is uploaded to aninformation system that is accessible by at least one of the patient, arepresentative of the patient, a doctor, a surgical facility, anindependent tissue storage facility, and a tissue processing facility.

In certain methods of the present invention, a packaged processed tissue(autograft) may be stored at the surgical facility for some period oftime before it is re-implanted in the patient from which the tissue wascollected. In some methods, when the autograft is to be stored for along term it may be stored at between about −40° C. and about −86° C.,or for a short term (e.g., less than 6 months) at between about −20° C.and −40° C. Depending on the processed tissue, it may be stored at roomtemperature. For example, a plasticizer may be added to the tissueduring or after the processing step which may allow the processed tissueto be stored at room temperature.

In certain aspects of the present invention, processing may includetreating the tissue with a plasticizer (i.e., glycerol, among others).Plasticizers may be added to the solution for temporary storage duringor immediately after treatment of the tissue, but prior to packaging thetissue for shipping, in some aspects of the present invention.Plasticizers may also be employed during or after processing of thetissue for storage of the tissue prior tore-implantation.

Plasticizers may be employed at concentrations in the range of fromabout 10% to 100% by weight or volume. Depending on the tissue or organbeing plasticized, the present plasticizers may be employed atconcentrations in the range of from about 13% v/v to about 100% v/v,from about 50% v/v to about 100% v/v, from about 60% v/v to about 100%v/v, from about 75% v/v to about 100% v/v, from about 10% v/v to about25% v/v, from about 10% v/v to about 20% v/v, from about 75% v/v toabout 90% v/v, from about 75% v/v to about 85% v/v, from about 80% v/vto about 85% v/v, or from about 75% v/v to about 85% v/v of the totalvolume. As an example, the concentration of plasticizer used toplasticize bone may be greater than about 70% v/v, greater than about75% v/v, from about 70% to about 90% v/v, from about 75% to about 80%v/v, about 70% v/v, or about 77% v/v.

In certain aspects of the present invention, during processing or at thetime a processed autograft is re-implanted, a gel, a paste, a putty, ora sponge that may aid in healing and union, may be applied to a surfaceand/or an edge of the graft or at the edges of a surgical implantationsite. Such gels, pastes, putties, and sponges may contain allograftmaterials (i.e., demineralized bone powder, and collagen, among others)and/or synthetic materials (i.e., polymers, calcium phosphate,hydroxyapatite, among others). Exemplary gels, pastes, and putties thatmay be employed in certain aspects of the present invention are soldunder the trade names of Grafton® (Osteotech, Inc., Eatontown, N.J.),DBX® (Musculoskeletal Transplant Foundation), Plexur™ Biocomposites(Osteotech, Inc., Eatontown, N.J.), BioSet™ (RTI Biologics, Alachua,Fla.), and Optium DBM® (LifeNet Health, Virginia Beach, Va.), amongothers. Exemplary compositions are also described in U.S. Pat. Nos.7,019,192; 7,001,430; 6,911,212, 6,458,375; 6,437,018; 6,326,018;6,030,635; and 5,702,677, among others known in the art. Collagensponges may also be used with a re-implanted autograft, in certainembodiments. In some aspects of the present invention, a biocompatibleadhesive (i.e., fibrin-based adhesives and collagen gels, among others)may be used to help hold an autograft in place upon re-implantation.

Certain aspects of the present invention are directed to an autologoustissue collection kit for use in collecting a tissue from a patient.Some autologous tissue collection kits of the present inventionincludes, (1) an instruction sheet containing at least a contact phonenumber for a tissue processing facility, a description of autologoustissue collection kit use, and a description of a method of packaging acollected tissue; (2) at least one label including a space for apatient's identifying information to be entered; (3) a sealable tissuecontainer; (4) a shipping container; and (5) at least one of a treatmentcomposition and an electronic storage device for storing at least apatient identification number. In some aspects of the present invention,the sealable tissue container in an autologous tissue collection kit maybe rigid and configured to fit directly into a centrifuge at a tissueprocessing facility. Some autologous tissue collection kits of thepresent invention may include sterile materials for packaging a tissuethat is collected, and the materials may be drapes or bags, amongothers. Certain tissue collection kits of the present invention maycontain forms to be filled out on-site when a tissue is collected.

As an example, a kit of the present invention may include (1) acontainer for holding or storing the tissue after removal from patientat the surgical facility; (2) a treatment solution, such as Allowash®,for cleaning and/or disinfecting the tissue at the surgical facility toreduce the bio-burden before processing; (3) temporary storage andshipment solution, for example, a solution containing plasticizer (andoptionally a decontaminating agent) which enables the tissue to beshipped at room temperature and eliminates the need to freeze the tissueprior to packaging the tissue for shipping; (4) a device for adding aninstrument insertion site to the graft for ease of maneuvering thetissue; (5) a microchip for tracking the tissue; (6) a container forstorage at the surgical facility and/or shipment to the processingfacility; and (7) a cooler comprising a compartment for storing theautologous tissue and other compartments for storing other accessories.The kit may include more than one of each of these components. Thepresent invention also provides methods for using the kit disclosedherein to procure and process autologous tissues for re-implantation ina patient.

The temporary storage and shipment solutions included in the kit may bebacteriostatic and/or bactericidal. The solutions may be a compositioncontaining a disinfectant. The storage and shipment solutions mayinclude a bacteriostatic agent and/or a bactericidal agent. The storageand shipment solutions may also include a preservation agent orplasticizer. The plasticizer may be glycerol. The plasticizer eliminatesthe need to ship and store the tissue at low temperature. As an example,the storage and shipment solutions may contain glycerol.

The device included in the kit allows the surgeon to drill one or morehole in the graft and to make screw threads. A syringe connector andtubing can be connected to the hole and used for flushing the treatmentsolution and rinsing solution through the tissue in the operating roomand later at the processing facility. The microchip may contain thepatient information and for documenting the treatment and processingconditions of the tissue.

The container included in the kit may have one or more openings or portsfor liquids to enter and exit and an opening for placing the tissue intothe container. The container may be deformable, nondeformable, or acombination thereof in which the container has a non-deformable exteriorand a deformable interior. The container may be made of material thatenables it to be deformable, or nondeformable or a combination thereof.The container may have an inlet opening and an outlet opening forrinsing solutions, treatment solutions, and storage solutions to enterand exit. The opening on the container for placing the tissue into orfor removal of the tissue out of the container may be closed with azipper or with a clamp. The process of cleaning, disinfecting, treating,storing, and/or shipping of the graft may all be performed in onecontainer. The container may be a bag or may contain a bag with thefeatures described above for the container.

In some aspects of the present invention, a collected tissue may bestored and processed in a processing container. In certain aspects, theprocessing container comprises at least one fluid (e.g., liquid or gas)inlet and one fluid outlet. The processing container may be large enoughto contain a collected tissue and a treatment or processing composition.In some aspects of the present invention, the processing container ismade of a material that does not react with the treatment composition(s)and/or processing composition(s), and can withstand centrifugation,and/or application of a vacuum. In certain aspects, the processingcontainer remains undamaged, when its contents (i.e., bone tissue and atreatment solution, among others) are subjected to ultrasonication,cavitation, and/or high pressure air bubbles. In some aspects of thepresent invention, the processing container is deformable (i.e., aplastic bag, among others). In certain aspects of the present invention,a deformable processing container may adhere to the surfaces of thecollected tissue during processing (i.e., when drawing a cleaningsolution through the tissue using a vacuum) improving fluid penetrationof the collected tissue.

In some aspects of the present invention, an autologous tissuecollection kit may further include a tool for creating an instrumentinsertion site in a tissue. Certain autologous tissue collection kits ofthe present invention may further include a surgical instrument forre-implanting an autograft. In certain aspects of the present invention,the tool for creating an instrument insertion site in an autograft andthe surgical instrument for re-implanting an autograft are the same. Insome aspects of the present invention, the tool is used to create athreaded hole that is used as an instrument insertion site in a tissue.

Some autologous tissue collection kits of the present invention mayinclude a treatment (or pre-treatment) composition for treating thetissue prior to processing, and the treatment composition may include atleast one of a decontaminating agent (such as a bactericidal agent), abacteriostatic agent, an enzyme, and a surfactant. The treatmentcomposition may include glycerol or another plasticizer in certainaspects of the present invention. The treatment composition may includea decontaminating agent and a plasticizer in some aspects of the presentinvention.

A shipping container of certain autologous tissue collection kits of thepresent invention may include a compartment for holding patient records,a compartment for holding the sealable tissue container, and acompartment for holding at a treatment (or pretreatment) composition, amicrochip, or a combination thereof. In some aspects of the presentinvention, the compartment for holding a sealable tissue container in ashipping container may be temperature controlled. Certain shippingcontainers used in the present invention may include labeling thatindicates that the container holds biohazardous and/or human tissue. Ashipping container may contain a donor identification, a patient'smedical record number, name of a surgical facility where re-implantationof an autograft will take place, a record of the number of pieces ofautograft tissue in the shipping container, and storage requirements, insome aspects of the present invention.

Certain aspects of the present invention are directed to an autologousbone graft for re-implantation into a recipient including a piece ofbone that is autologous to the recipient, wherein the piece of boneincludes an instrument insertion site that is capable of securelycoupling to (1) a surgical placement instrument and (2) at least one ofa solution injector and a solution evacuator. In some autologous bonegrafts of the present invention, the instrument insertion site may bethreaded. In some aspects of the present invention, the solutioninjector may be a syringe and the instrument insertion site acts as asyringe connection port. In certain aspects, the solution injector maybe a pump that pumps a solution into an autologous bone graft throughthe instrument insertion site. Alternatively, a solution evacuator maybe a pump that pumps (evacuates) a solution out of an autologous bonegraft through the instrument insertion site. A solution that is injectedor evacuated from an autograft may be a processing/treatment solution, astorage solution, or a rinse solution, among others, in some aspects ofthe present invention.

Certain autologous bone grafts of the present invention are prepared bya cleaning and/or processing method known in the art (see discussionabove). In some aspects of the present invention, an autologous bonegraft is prepared by a processing method including at least one cleaningstep and at least one rinsing step. In certain aspects, a tissue thatwas collected from the patient may be processed using proprietaryAllowash® technology of LifeNet Health, Virginia Beach, Va. in preparingan autologous bone graft of the present invention. In certain aspects ofthe present invention, an autologous bone graft is prepared by aprocessing method that includes at least one of perforating the bonegraft, demineralizing the bone graft, applying a bioactive factor to thebone graft, and seeding the bone graft with viable cells. In certainaspects of the present invention, an autologous bone graft furtherincludes at least one of an allograft tissue, a synthetic implantmaterial, and a biocompatible adhesive (as discussed above) that may aidin at least one of wound healing, union of the graft with tissue at theimplant site, and keeping the graft in place until union of tissuesoccurs.

Some autologous bone grafts of the present invention may be sterile.Certain autologous bone grafts of the present invention may be preparedfrom a tissue that had previously been contaminated with a bacterium, asingle-celled eukaryote, or a virus. Some aspects of the presentinvention are directed to a packaged autologous bone graft having asterility assurance level of at least about 10-6. In certain aspects ofthe present invention, a packaged autograft may be marked with anexpiration date. An autograft may be packaged in a double peel packpouch in some aspects of the present invention, and in certain aspectsthe double peel pack pouch may be validated to maintain integrity for atleast 5 years.

The present invention may be used to pre-treat and process organs, aswell as tissues, surgically removed from a patient. The organs afterpre-treatment at the surgical facility may be packaged for shipping to aprocessing facility. After processing, the organs may be may be storedat the processing facility or packaged for shipping to the processingfacility for re-implantation.

In some aspects of the invention, the tissue may be removed from asubject. The subject may be a patient. The patient may be an animal. Theanimal may be a mammal. The mammal may be a human, a cow, a pig, a goat,a dog, a horse, or a sheep.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the claimed invention. Thefollowing working examples therefore, specifically point out embodimentsof the present invention, and are not to be construed as limiting in anyway the remainder of the disclosure. All articles, publications, patentsand documents referred to throughout this application are herebyincorporated by reference in their entirety.

EXAMPLES Example 1

A right decompressive craniectomy was performed on a 43-year-old patientafter a motor vehicle collision. The bone flap removed during thecraniectomy was stored in a freezer at −80° C. On the 70th day after theinjury, after the patient had recovered, surgeons performed acranioplasty, using the stored bone flap to close the defect from theprevious craniectomy. A week later, the patient presented with clinicalsymptoms of infection, including running a high fever. The cranial boneflap was removed, the patient was treated for the infection, and thebone flap was sent to LifeNet Health for processing. The bone flap wasswabbed for contaminants during surgery and prior to processing atLifeNet Health, and Enterobacter cloacae were cultured from the swabs.(Swab cultures were tested for contaminating agents using boththioglycolate and trypticase soy broth media in a two-temperature/14-dayculture protocol.) The bone flap was then cleaned and disinfected usingprocesses that included LifeNet Health's proprietary Allowash®technology. Following processing, swab cultures taken from the bone flapwere negative. Subsequently, the bone flap was treated with acontrolled, low dose (i.e., 18 kGy to 58 kGy minimum absorbed dose) ofgamma irradiation at low temperatures (i.e., −20° C. to −50° C.). Thecleaned and irradiated bone flap was re-implanted in the patient afterthree months. There has been no recurrence of infection at the defectsite, since the bone flap was re-implanted.

Pictures of the bone flap before and after processing at LifeNet Healthare shown in FIGS. 1A and 1B, respectively.

Example 2

A left decompressive craniectomy was performed on a 20-year-old patientafter trauma at Hospital A. The bone flap removed during the craniectomywas stored in a freezer at −80° C. Following his recovery, the patientwas transferred to Hospital B for cranioplasty. The bone flap wastransported to Hospital B on dry ice, but arrived at room temperature,and it was deemed unsafe for re-implantation. Hospital B stored the boneflap in its freezer at −80° C., until it could be sent to LifeNet Healthfor processing. At LifeNet Health, the bone flap was swabbed forcontaminants, and Staphylococcus epidermis was cultured from the swabs.(Swab cultures were tested for contaminating agents using boththioglycolate and trypticase soy broth media in a two-temperature/14-dayculture protocol.) The bone flap was then cleaned and disinfected usingprocesses that included LifeNet Health's Allowash® technology. Followingprocessing, swab cultures taken from the bone flap were negative.Subsequently, the bone flap was treated with a controlled, low dose(i.e., 18 kGy to 58 kGy minimum absorbed dose) of gamma irradiation atlow temperatures (i.e., −20° C. to −50° C.). The cleaned and irradiatedbone flap was re-implanted in the patient seven months later. There hasbeen no recurrence of infection at the defect site, since the bone flapwas re-implanted. Pictures of the bone flap before and after processingat LifeNet Health are shown in FIGS. 2A and 2B, respectively.

Example 3

A craniectomy was performed during excision of a tumor in an11-year-old, immune-compromised patient. The patient wasimmune-compromised due to tumor therapy. The bone flap was sent toLifeNet Health for processing. The bone flap was swabbed forcontaminants, and Staphylococcus aureus and Streptococcus viridans werecultured from the swabs. (Swab cultures were tested for contaminatingagents using both thioglycolate and trypticase soy broth media in atwo-temperature/14-day culture protocol.) The bone flap was then cleanedand disinfected using processes that included LifeNet Health's Allowash®technology. Following processing, swab cultures taken from the bone flapwere negative. Subsequently, the bone flap was treated with acontrolled, low dose (i.e., 18 kGy to 58 kGy minimum absorbed dose) ofgamma irradiation at low temperatures (i.e., −20° C. to −50° C.). Thecleaned and irradiated bone flap was re-implanted in the patient sixweeks later. There have been no complications reported, since the boneflap was re-implanted. Pictures of the bone flap before and afterprocessing at LifeNet Health are shown in FIGS. 3A and 3B, respectively.

1. A method of preparing a clean, disinfected tissue implant forimplantation, the method comprising: a) pre-treating a surgicallyremoved piece of tissue from a subject with at least one treatmentcomposition to produce a pre-treated tissue; b) shipping the pre-treatedtissue to a tissue processing facility; c) receiving the pre-treatedtissue at the tissue processing facility; d) processing the pre-treatedtissue to clean and disinfect the tissue; e) storing the cleaned anddisinfected tissue at a storage facility; and f) providing a package toa surgical facility comprising the cleaned and disinfected tissue and atracking device, wherein the tissue further comprises viable cells, andwherein the tissue is bone, cartilage, or soft tissue.
 2. The method ofclaim 1, wherein the tracking device comprises a microchip.
 3. Themethod of claim 1, wherein the tracking device comprises a thumb drive.4. The method of claim 1, wherein the bone is mandible bone, frontalbone, parietal bone, sphenoid bone, temporal bone or nasal bone.
 5. Themethod of claim 1, wherein the bone is a bone of the hand, a bone of thefoot, a bone of the arm, or a bone of the leg.
 6. The method of claim 1,wherein the bone is a tibial bone or a femoral bone.
 7. The method ofclaim 1, wherein the soft tissue is at least one of a ligament, atendon, a fascia, or a muscle.
 8. The method of claim 1, furthercomprising packaging the cleaned and disinfected tissue afterprocessing.
 9. The method of claim 1, wherein the pre-treating comprisestreating the collected tissue with an antibiotic solution, abactericidal solution, a virucidal, a glycerol-containing solution,water, saline or a combination thereof.
 10. The method of claim 1,wherein the storing comprises including the tracking device.